Recombinant Expression Of Aspergillus Niger Derived Pep In Non-Spore A.niger SH-2

Prolyl endoprotease(PEP) derived from Aspergillus niger is an enzyme that couldpreferentially hydrolyse protein and peptide at the carboxyl side of proline residues. PEPcould be used in preveting chill-haze in beer, treating celiac sprue and debittering of proteinhydrolysates. Customers of our coutry have no choice but to use PEP products imported.Filamentous fungi are able to secret so large amout of hydrolase enzymes that are widely usedas factory cell to express homologous and heterologous enzymes. As GRAS microorganism,A.niger is deemed safe, productive and high-quality to express target protein.A.niger used as host in this study is a sporeless glucoamlase producer whose hypha isshort but sturdy. A high throughput screening method for productive PEP transformants wasinvented, at the same time6different PEP expression frame was constructed and transferredto the wild non-spore A.niger through PEG-mediated protoplast transformation. Finally, themost productive PEP expression frame was selected. The experiments data shows that6different PEP expression frame was successfully inserted into the gemoe of non-spore A.nigerand the recombinate PEP was secreted. The most productive PEP expression frame wasconformed through the high throughput screening of283PEP positive transformantsharboring one of the six PEP expression frame.The total amout of protein secreted by wild non-spore A.niger SH-2is as much as30g/L,which suppress the the secretion, downstream purification and formulation of recombinateprotein. A high efficient multigene knock-out strain was constructed through knockout ofpyrG and kusA genes of non-spore A.niger SH-2, which makes afterward multi-genesknockout possible and efficient. Finally the optimized PEP expression frame was transferredinto the Δ pyrG+Δ kusA strain and the most productive transformants was fermented in the5L fermentation tank. The PEP enzyme activity of the supernatant is1.81nkat/ml, relativelyvery potential campared to DSM’ PEP concentration Brewer Clarex whose enzyme activity is11.7nkat/ml.